This invention relates to inhibitors for dipeptidyl peptidase IV which are used to regulate substrate activity. As used herein, the term “substrate” denotes chemokines, cytokines and biological peptides which are substrates for DPP-IV. This invention also relates to the use of DPP-IV inhibitors in the treatment of medical disorders which may result from the inactivation of substrates implicated in the medical disorder. As used herein, dipeptidyl peptidase IV is alternatively described as “DPP-IV,” “DP-IV” and “CD26.” CD26 is an ectoenzyme with activity identical to that of DPP-IV.
DPP-IV is a serine type exopeptidase with high substrate specificity cleaves N-terminal dipeptides from proteins if the penultimate amino acid is proline, or in some cases alanine (Fleischer, B. Immunol. Today 15:180 (1994)).
A class of low molecular weight synthetic monomeric molecules with high affinity for CD26 have previously been developed and characterized (G. R. Flentke et al., Inhibition of dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro dipeptides and use of these inhibitors to examine the role of DP-IV in T-cell function, PNAS (USA) 88, 1556-1559 (1991); W. G. Gutheil and W. W. Bachovchin, Separation of L-Pro-DL-boroPro into Its Component Diastereomers and Kinetic Analysis of Their Inhibition of Dipeptidyl Peptidase IV: A New Method for the Analysis of Slow, Tight-Binding Inhibition, Biochemistry 32, 8723-8731 (1993)). These molecules have been shown to be potent and specific synthetic inhibitors for CD26-associated DP-IV proteinase activity.
Representative monomeric structures of these transition-state-analog-based inhibitors, Xaa-boroPro, wherein Xaa is an amino acid residue, include Pro-boroPro, Ala-boroPro, Val-boroPro, and Lys-boroPro. BoroPro refers to the analog of proline in which the carboxylate group (COOH) is replaced with a boronyl group [B(OH)2]. Pro-boroPro, the most thoroughly characterized of these inhibitors, has a Ki of 16 picomolar (pM) (W. G. Gutheil and W. W. Bachovchin, supra). Val-boroPro has an even higher affinity, with a Ki of 1.6 pM (W. G. Gutheil and W. W. Bachovchin, supra; R. J. Snow et al., Studies on Proline Boronic Acid Dipeptide Inhibitors of Dipeptidyl Peptidase IV: Identification of a Cyclic Species Containing a B—N Bond, J. Am. Chem. Soc. 116, 10860-10869 (1994)). Thus, these Xaa-boroPro inhibitors are about 10+6 fold more potent than the next best known inhibitors.
U.S. Pat. No. 4,935,493 (Bachovchin '493) and U.S. Pat. No. 5,462,928 (Bachovchin '928), both of which are incorporated herein by reference, disclose protease inhibitors and transition state analogs (the '493 patent) and methods for treating transplant rejection in a patient, arthritis, or systemic lupus erythematosis (SLE) by administering a potent inhibitor of the catalytic activity of soluble amino peptidase activity of dipeptidyl peptidase type IV (G. R. Flentke et al., supra).
Chemokines, or chemoattractant cytokines, are a family of small proteins with a conserved cysteine motifs. These small proteins have been implicated in a wide range of disease states, such as acute and chronic inflammatory processes, angiogenesis, leukocyte migration, regulation of cell proliferation and maturation, hematopoiesis, viral replication, and other immunoregulatory functions. Chemokines are expressed by a number of different cells and have distinct but overlapping cellular targets.
There are two groups of chemokines defined according to their structural characteristics: the CXC and the CC groups. In addition, C-chemokines and CX3C-chemokines have been identified. Members of the CXC group, which include SDF-1 and IL-8, attract mostly neutrophils, while the CC group acts on monocytes and granulocytes. The CC group includes such molecules as human monocyte chemotactic protein 1 (MCP-1) and RANTES. MCP-1 and RANTES are potent direct mediators of the release of histamine by human basophils. Both groups of chemokines are involved in lymphocyte migration to inflammatory sites.
The majority of chemokine receptors are transmembrane spanning molecules which belong to the family of G-protein-coupled receptors. Many of these receptors couple to guanine nucleotide binding proteins to transmit cellular signals. Chemokines and receptor expression is upregulated during inflammatory responses and cellular activation. Chemokines, through binding to their respective receptors, have been shown to be involved in a number of physiologic conditions. For instance, chemokines of the CXC group, like Interleukin-8, can stimulate angiogenesis, while Platelet Factor-4, growth-related oncogene-β (GRO-β) and interferon-γ induced Protein-10 (IP-10) inhibit endothelial cell proliferation and angiogenesis. Interleukin-8 stimulates endothelial cell proliferation and chemotaxis in vitro, and appears to be a primary inducer of macrophage induced angiogenesis. It was shown that the activities of these chemokines are dependent on the NH2-terminal amino acid sequence (Streiter et al., J. Biol. Chem., 270; pages 27348-27357). SDF-1, another CXC chemokine, is active in the recruitment and mobilization of hematopoietic cells from the bone marrow, as well as the attraction of monocytes and lymphocytes. In addition, it interferes with cellular infection of HIV-1 by blocking the interaction of HIV-1 with CXCR-4. As with other chemokines in this group, the amino terminal sequence regulates its activity (Shioda, T., et al., PNAS, 95; pages 6331-6336).
Chemokine receptors have been shown to serve not only as receptors to chemokines, but most recently have been identified as receptors for a variety of microbes and the HIV-1 virus. For instance, the Duffy blood group Ag, a chemokine receptor on erythrocytes, is the receptor for the malaria parasite Plasmodium vivax, and the platelet activating receptor is a receptor for Streptococcus pneumonia. 
A number of chemokines, such as RANTES, MIP-1 and SDF-1, or cytokines like IL-2, or peptides like GLP-1, GLP-2, and Substance P, are substrates for DPP-IV. DPP-IV cleaves peptides at the NH2 terminus if the penultimate amino acid is proline. Several cytokines and chemokines have the conserved sequence NH2X-Pro-X, and have been shown to be substrates for DPP-IV. DPP-IV is expressed on the surface of T cells and macrophages. The relationship of CD26 protease activity to its immune function is not clear, however there are indications that cleavage by CD26 of the NH2-dipeptide of several cytokines changes their receptor specificity and/or their functional activities.
Cytokines that are known to be potential substrates for DPP-IV include G-CSF, erythropoietin, IL-1β, IL-2, IL-3, IL-6, IL-11, TNF-β and GM-CSF.
In addition to cytokines and chemokines, a number of biologically active peptides have, on their amino termini, the amino acid sequence Xaa-Pro-Xaa, which serves as the substrate for DPP-IV. Among these are the Glucagon Like Peptides, GLP-1 and GLP-2. GLP-1 is involved in insulin release and glucose uptake, and cleavage by DPP-IV causes inactivation of its activity. Inhibition of DPP-IV will result in the prolonged activation state of this peptide, and represents a therapeutic indication of DPP-IV inhibitors. GLP-2 peptide is involved in intestinal growth and nutrient uptake, and increased activity of GLP-2 will result in an increased nutrient uptake for individuals with intestinal diseases.
In addition to the foregoing, CD26 is known to be highly expressed on hepatosplenic T cell lymphoma, and DPP-IV activity, or the ability of CD26 to bind to collagen fibronectin on the extracellular matrix, may be part of the pathogenic mechanism utilized in by neoplastic cells (Ruiz et al., Cytometry 1998, 34: pages 30-35).
DPP-IV and DPP-II levels are known to be significantly increased in the gingival crevicular fluid of patients with periodontitis. Increased levels of these two proteases seem to be associated with increased attachment loss. It is believed that collagen-CD26 interactions are a part of the pathological observations.
PCT published application WO 95/11689 discloses the use of inhibitors of DPP-IV to block CD26, thereby blocking entry of HIV into CD26-bearing cells. These inhibitors are tetrapeptides having the general formula X-Pro-Y-boroPro where X and Y are chosen from any amino acid. Although the dipeptides are also disclosed, the reference states that dipeptides are unstable, and tetrapeptides are preferred. The inhibitors are used to treat pre-symptomatic HIV-infected patients not by neutralizing the virus, but by blocking viral entry into the cells. The reference does not disclose the effect of DPP-IV inhibition on chemokine activity.